In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the specimen. The focus of the illuminating light beam is moved by means of a controllable beam deflection device (generally by tilting two mirrors) in one specimen plane; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X and the other in the Y direction. Tilting of the mirrors is brought about, for example, with galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position. Also known, in addition to these so-called “beam scanning” methods, are scanning microscopes having a spatially stationary illuminating light beam, in which the specimen is moved by means of a precision positioning stage for scanning. These are called “specimen scanning” microscopes.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a pinhole (called the excitation pinhole), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen arrives via the beam deflection device back at the beam splitter, passes through it, and is then focused onto the detection pinhole, behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which, by sequential scanning of the specimen, results in a three-dimensional image. Usually a three-dimensional image is achieved by acquiring image data in layers.
The power level of the light coming from the specimen is measured at fixed time intervals during the scanning operation, and thus sampled one grid point at a time. The measured value must be unequivocally associated with the relevant scan position so that an image can be generated from the measured data. Advantageously, the status data of the adjusting elements of the beam deflection device are also continuously measured for this purpose, or—although this is less accurate—the setpoint control data of the beam deflection device are used directly.
In a transmitted-light arrangement, for example, it is also possible to detect the fluorescent light or the transmission of the exciting light on the condenser side. The detected light beam then does not pass via the scanning mirror to the detector (non-descan configuration). In the transmitted-light configuration, a condenser-side detection pinhole would be necessary for detection of the fluorescent light in order to achieve three-dimensional resolution as in the descan arrangement described. In the case of two-photon excitation a condenser-side detection pinhole can be dispensed with, however, since the excitation probability is a function of the square of the photon density (proportional to [intensity]2), which of course is much higher at the focus than in the adjacent regions. The great majority of the fluorescent light to be detected therefore derives with high probability from the focus region, which makes superfluous any further differentiation, using a pinhole arrangement, between fluorescent photons from the focus region and fluorescent photons from the adjacent regions.
The resolution of a confocal scanning microscope is defined, inter alia, by the intensity distribution and the spatial extension of the focus of the exciting light beam. An arrangement for increasing the resolution of a confocal scanning microscope for fluorescent applications is known from PCT/DE/95/00124. Here the lateral edge regions of the focus volume of the exciting light beam are illuminated with a light beam of a different wavelength (called the “stimulating light beam”) that is emitted by a second laser, so that the specimen regions excited there by the light of the first laser are brought back to the ground state in stimulated fashion. Only the light spontaneously emitted from the regions not illuminated by the second laser is then detected, so that the overall result is an improvement in resolution. The term “stimulated emission depletion” (STED) has become established for this method.
A new development has shown that a resolution improvement can be simultaneously achieved both laterally and axially if the focus of the stimulating light beam can be made internally hollow. This is done by introducing into the beam path of the stimulating light beam a round λ/2 plate which has a diameter smaller than the beam diameter and is therefore overilluminated.
Microscopes for STED microscopy are very complex and difficult to align, since the focus of the exciting light beam must always have a fixed spatial relationship to the focus of a stimulating light beam. This problem becomes very particularly difficult in beam-scanning systems, since in these systems the foci of the exciting light beam and the detected light beam must be guided over or through the specimen simultaneously and in stationary fashion with respect to one another.
Scanning microscopes for STED microscopy that are implemented on an optical bench are very bulky and, because of their size, very difficult to protect against external influences such as mechanical vibrations or environmental temperature fluctuations. For this reason, only specimen-scanning systems have so far been implemented. Because of the complexity involved, scanning microscopes cannot be converted for STED microscopy by retrofitting conventional scanning microscopes.